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IDENTIFICATION and CHARACTERIZATION of Gatase1-LIKE Arac-FAMILY TRANSCRIPTIONAL REGULATORS in BURKHOLDERIA THAILANDENSIS

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IDENTIFICATION and CHARACTERIZATION of Gatase1-LIKE Arac-FAMILY TRANSCRIPTIONAL REGULATORS in BURKHOLDERIA THAILANDENSIS University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2018 IDENTIFICATION AND CHARACTERIZATION OF GATase1-LIKE AraC-FAMILY TRANSCRIPTIONAL REGULATORS IN BURKHOLDERIA THAILANDENSIS. Adam Michael Nock University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Microbiology Commons Recommended Citation Nock, Adam Michael, "IDENTIFICATION AND CHARACTERIZATION OF GATase1-LIKE AraC-FAMILY TRANSCRIPTIONAL REGULATORS IN BURKHOLDERIA THAILANDENSIS." (2018). Graduate College Dissertations and Theses. 903. https://scholarworks.uvm.edu/graddis/903 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. IDENTIFICATION AND CHARACTERIZATION OF GATASE1-LIKE ARAC- FAMILY TRANSCRIPTIONAL REGULATORS IN BURKHOLDERIA THAILANDENSIS. A Dissertation Presented by Adam Michael Nock to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Microbiology and Molecular Genetics May, 2018 Defense Date: March 14, 2018 Dissertation Examination Committee: Matthew J. Wargo, Ph.D., Advisor John W. Barlow, D.V.M., Ph.D., Chairperson Jason W. Botten, Ph.D. Mary J. Dunlop, Ph.D. Keith P. Mintz, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT The ability of bacteria to detect their surroundings and enact an appropriate response is critical for survival. Translation of external signals into a coherent response requires specific control over the transcription of DNA into RNA. Much of the regulation at this step is accomplished by transcriptional regulators, proteins that bind to DNA and alter gene expression. A wide-spread variety of regulators in bacteria is the AraC-family. These regulators are divided into two conserved domains and respond to a variety of compounds owing to different N-terminal domains. A subfamily of these regulators, GATase1-like AraC-family transcriptional regulators (GATRs), is described. These proteins contain an N-terminal domain with structural characteristics similar to enzymes that synthesize amine-containing compounds. Members of this subfamily of transcriptional regulators are found in a wide range of bacteria, however, few are characterized. A relatively high number of GATRs are encoded in the Burkholderia thailandensis genome. Therefore, we utilized this bacterium as a model to explore the function and diversity of these regulators. GATRs in B. thailandensis divided into two groups based on bioinformatics analysis. The first group includes three members which we identified that contribute to the positive regulation of glycine betaine (GB) catabolism. GB can be utilized as a nutrient source or as a potent osmoprotectant. The regulation of this pathway in B. thailandensis differs from previously established models due to the interplay of these regulators. Homologs of two other GATRs in this group were identified that regulate carnitine and arginine catabolism. The second group of GATRs contains uncharacterized members with no known functions. A genetic strategy for engineering constitutive GATRs was developed and employed to investigate the transcriptional regulons of these GATRs. This approach yielded the identification of a novel GATR that represses expression of an operon producing a formaldehyde detoxification system, and is the first example of a GATR that functions as a repressor. CITATIONS Material from this dissertation has been published in the following form: Nock A.M., Wargo M.J.. (2016). Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators. Journal of Bacteriology, 198(18): 2503-14. ii ACKNOWLEDGEMENTS Any success that I have encountered, I must credit to the good fortune of a string of invaluable mentors. Although too long a list to properly enumerate, two in particularly must be mentioned here. Sohail Malik was instrumental in my training and essentially everything I know about biochemistry can be attributed directly to his instruction. His tutelage instilled both an appreciation for precise science and an orderly workspace. Sohail’s attitude concerning science is as much curiosity as seriousness, and well- reflected the overall atmosphere at Rockefeller University, which is to continue the search for understanding the world around us. And without the guidance Thomas Montville at Rutgers, I would likely not have made it to this crossroads at all. Tom’s lab solidified my interest in microbes while simultaneously helping me right my academic career and build a strategy to achieve future goals. My parents James and Julia sacrificed a great deal of their comfort and interests in order to secure a better education and experience for John, Sarah, and myself. My wife Audra has provided love and support throughout this venture. Her parents, Ben and Grace, as well as our extended family, have been a continuous font of encouragement. And in this time, somehow my dearest friends have not forgotten me no matter the distance. The generosity of the people in my life has been humbling. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................. iii LIST OF FIGURES ......................................................................................................... ix LIST OF TABLES ........................................................................................................... xi CHAPTER 1: INTRODUCTION ..................................................................................... 1 1.1 Sensing and responding to the environment ................................................... 1 1.2 Glycine betaine as a metabolite and signal ..................................................... 2 1.3 The AraC family of transcriptional regulators ................................................ 3 1.4 Glutamine amidotransferase 1 (GATase 1)-containing AraC family transcriptional regulators (GATRs) ..................................................................... 6 1.5 Burkholderia thailandensis as a model organism ........................................... 9 1.6 Focus of study and approach......................................................................... 14 1.7 Chapter 1 Figures .......................................................................................... 16 1.8 Chapter 1 References .................................................................................... 28 CHAPTER 2: CHOLINE CATABOLISM IN BURKHOLDERIA THAILANDENSIS IS REGULATED BY MULTIPLE GLUTAMINE AMIDOTRANSFERASE1- CONTAINING ARAC-FAMILY TRANSCRIPTIONAL REGULATIORS ................ 36 2.1.1 Abstract ...................................................................................................... 37 2.1.2 Importance ................................................................................................. 38 2.2 Introduction ................................................................................................... 38 2.3 Methods......................................................................................................... 40 2.3.1 Culture conditions ................................................................................. 40 iv 2.3.2 Strain Construction ............................................................................... 40 2.3.3 Growth assays ....................................................................................... 41 2.3.4 Alignments and phylogenetic tree construction .................................... 42 2.3.5 RNA-Seq ............................................................................................... 42 2.3.6 qRT-PCR to confirm RNA-Seq findings .............................................. 44 2.3.7 Purification of GbdR1, GbdR2, and SouR............................................ 44 2.3.8 Electrophoretic mobility shift assays (EMSA) .................................... 46 2.3.9 Generation of reporter constructs and β-galactosidase assays .............. 46 2.4 Results ........................................................................................................... 47 2.4.1 Organization of predicted B. thailandensis orthologs of P. aeruginosa choline catabolic genes .......................................................................... 47 2.4.2 gbdR1 and gbdR2 contribute differentially to choline catabolism ........ 49 2.4.3 GbdR1 and GbdR2 regulate transcription of genes involved in the choline catabolic pathway .............................................................................. 50 2.4.4 The glyA promoter is induced by choline under the control of GbdR1 and GbdR2 ...................................................................................................... 52 2.4.5 GbdR1 and GbdR2 bind directly to the glyA promoter ........................ 54 2.4.6 B. thailandensis SouR regulates sarcosine metabolism ........................ 55 2.5 Discussion ..................................................................................................... 57 2.6 Acknowledgements ....................................................................................... 62 2.7 Chapter 2 Figures
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